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FOXM1-mediated regulation of reactive oxygen species and radioresistance in oral squamous cell carcinoma cells

ABSTRACT: Radioresistance is a major obstacle to the successful treatment of oral squamous cell carcinoma (OSCC). To help overcome this issue, we have developed clinically relevant radioresistant (CRR) cell lines, generated by irradiating parental cells over time, which are useful for OSCC research. In the present study, we conducted gene expression analysis using CRR cells and their parental lines to investigate the regulation of radioresistance in OSCC cells. Based on gene expression changes over time in CRR cells and parental lines subjected to irradiation, forkhead box M1 (FOXM1) was selected for further analysis in terms of its expression OSCC cell lines, including CRR cell lines, and clinical specimens. We suppressed or upregulated the expression of FOXM1 in OSCC cell lines, including CRR cell lines, and examined radiosensitivity, DNA damage, and cell viability under various conditions. The molecular network regulating radiotolerance was also investigated, especially the redox pathway, and the radiosensitizing effect of FOXM1 inhibitors was examined as a potential therapeutic application. We found that FOXM1 was not expressed in normal human keratinocytes but was expressed in several OSCC cell types. The expression of FOXM1 was upregulated in CRR cells compared with that detected in the parental cell lines. In clinical specimens, FOXM1 expression was upregulated in cells that survived irradiation. FOXM1-specific siRNA treatment increased radiosensitivity, whereas FOXM1 overexpression decreased radiosensitivity, and DNA damage was altered significantly under both conditions as well as the levels of redox-related molecules and reactive oxygen species production. Treatment with the FOXM1 inhibitor thiostrepton had a radiosensitizing effect and overcame radiotolerance in CRR cells. According to these results, the FOXM1-mediated regulation of reactive oxygen species could be a novel therapeutic target for the treatment of radioresistant OSCC; thus, treatment strategies targeting this axis might overcome radioresistance in this disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE210922 | GEO | 2022/10/01

REPOSITORIES: GEO

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Project description:Background: Research using in vitro canine mammary carcinoma cell lines and studies involving naturally occurring canine mammary tumours are not only fundamental models used to advance the understanding of cancer in veterinary patients but are also regarded as excellent translational models for human breast cancer. In human medicine, following disease classification and staging breast cancer is typically treated with multimodal therapies including radiotherapy. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Comparative canine and human studies investigating the molecular mechanisms of radioresistance may lead to the development of effective cancer treatments that benefit both species. Methods: Human (MDA-MB-231, MCF-7 and ZR-751) and canine (REM-134) radioresistant cell lines were established by exposing parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Intrinsic differences between parental and radioresistant cells were investigated by whole-transcriptome gene expression analysis. Gene enrichment and pathway-focused analyses identified signalling networks differentially activated in radioresistant cells. Results: Colony formation assays identified a range of intrinsic radiosensitivities in the human and canine parental cell lines with the REM-134 cell line showing significantly greater radioresistance compared to the human cell lines. Colony formation and proliferation assays confirmed radioresistance in all developed radioresistant cell lines. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. Gene analysis identified that following the acquisition of radioresistance in MCF-7 and ZR-751 cell lines the cell lines changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. MDA-MB-231 and MDA-MB-231 RR cell lines both classified as basal, whereas the REM-134 and REM-134 RR cell lines with enriched for HER2 overexpression. Whole-transcriptome gene expression analysis identified down-regulation of ER signalling genes and up-regulation of genes associated with PI3K, MAPK and WNT pathway activity in radioresistant cell lines derived from ER+ cells; this was confirmed by western blot, which showed increased p-AKT and p-ERK expression following radiation. Conclusions: To our knowledge, our study is the first to develop a canine radioresistant cell line and provide a comparative genetic and phenotypic analysis of the mechanisms of acquired radioresistance between canine and human cell lines. The cellular process that occur with the development of acquired radioresistance are similar between the human and canine cell lines; this suggests that not only is the canine model an appropriate model to study human disease but that treatment strategies used in human medicine may also be applicable to veterinary patients.

Project description:Background: Radiotherapy plays an important role in the multimodal treatment of breast cancer. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Development of effective cancer treatments will require further molecular dissection of the processes that contribute to resistance. Methods: Radioresistant cell lines were established by exposing MDA-MB-231, MCF-7 and ZR-751 parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Intrinsic differences and changes in response to radiation between parental and radioresistant cells were investigated by whole-transcriptome gene expression analysis. Gene enrichment and pathway-focused analyses identified signalling networks differentially activated in radioresistant cells, which were confirmed by western blotting. Results: Proliferation and colony formation assays confirmed radioresistance. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition, and limited activation of DNA damage and apoptotic pathways in response to 2 Gy ionising radiation. Significantly, acquisition of radioresistance in MCF-7 and ZR-751 cell lines resulted in a loss of expression of both ERα and PgR and an increase in EGFR expression; based on gene analysis they changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. Whole-transcriptome gene expression analysis identified down-regulation of ER signalling genes and up-regulation of genes associated with PI3K, MAPK and WNT pathway activity in radioresistant cell lines derived from ER+ cells; this was confirmed by western blot, which showed increased p-AKT and p-ERK expression following radiation. Conclusions: This is the first study to date that extensively describes the development and characterisation of three novel radioresistant breast cancer cell lines through both genetic and phenotypic analysis. More changes were identified between parental cells and their radioresistant derivatives in the ER+ (MCF-7 and ZR-751) compared with the ER- cell line (MDA-MB-231) model; however, multiple and likely interrelated mechanisms were identified that may contribute to the development of acquired resistance to radiotherapy.

Project description:The molecular mechanisms underlying exceptional radioresistance in pancreatic cancer remain elusive. In the present study, we established a stable radioresistant pancreatic cancer cell line MIA PaCa-2-R by exposing the parental MIA PaCa-2 cells to fractionated ionizing radiation (IR). Systematic proteomics and bioinformatics comparison of protein expression in MIA PaCa-2 and MIA PaCa-2-R cells revealed that several growth factor- and cytokine-mediated pathways, including the OSM/STAT3, PI3K/AKT and MAPK/ERK pathways, were activated in the radioresistant cells, leading to enhanced cell migration, invasion and epithelial-mesenchymal transition (EMT), and inhibition of apoptosis. We focused functional analysis on one of the most upregulated proteins in the radioresistant cells, CD73, which is a cell surface protein that is overexpressed in a variety types of cancer. Ectopic overexpression of CD73 in the parent cells resulted in radioresistance and conferred resistance to IR-induced apoptosis. Knockdown of CD73 resensitized the radioresistant cells to IR and IR-induced apoptosis. The effect of CD73 on radioresistance and apoptosis is independent of the enzymatic activity of CD73. Further studies suggest that CD73 confers acquired radioresistance in pancreatic cancer cells at least in part through inactivating proapoptotic protein BAD via phosphorylation of BAD at Ser-136. Furthermore, we found that knockdown of CD73 in the radioresistant cells alone reverted the gene expression and phenotype of the radioresistant cells from those of mesenchymal-like cells to the ones of epithelial cells, demonstrating that CD73 upregulation is required for maintaining EMT in the radioresistant cells. Our results support the notion that the enhanced growth factor/cytokine signaling that promotes epithelial-mesenchymal plasticity, and acquisition of cancer stem-like cell properties contributes to acquired radioresistance in the residual surviving cells after fractionated irradiation, and that CD73 is a novel downstream factor of those enhanced signaling and acts to confers acquired radioresistance and maintains EMT in the radioresistant pancreatic cancer cells.

Project description:Pancreatic ductal adenocarcinoma is one of the most aggressive cancer types and is well-known for its general low intrinsic radiosensitivity. In order to resolve mechanisms of how PDAC cells carry out radioresistance, a combination of proteomics and phosphoproteomics of two radiosensitive as well as radioresistant murine PDAC cell lines upon exposition to radiation was performed. A high depth of (phospho)proteomic identifications with > 13000 confidently identified phosphorylation sites and > 7800 proteins was achieved. Measuring the response of the phosphoproteome upon radiation revealed >700 phosphorylation sites on >400 proteins which were regulated in all four cell lines independently of the sensitivity status. This analysis validated already known radiomarkers but also uncovered novel members of the radiation-dependent signaling network in PDAC cells that were shown to be exclusively based on protein phosphorylation but not protein expression. Among those radioresponsive phosphoproteins were ten novel ATM substrates, which were validated by in vitro kinase assays. Comparison of radiosensitive and -resistant cells revealed a complex regulation of apoptotic processes, while changes in expression of DNA repair proteins seemed to not play a pivotal role. Especially increased expression of NQO1 was found to be a potential mechanism enabling resistance by clearing harmful reactive oxygen species from the PDAC cells. Further analysis of the radioresistance-associated-phosphoproteome, which was especially enriched in phosphoproteins with cytoskeleton organizational function, uncovered increased Actin dynamics and FAK activity in radioresistant cells. Both likely lead to increased survival, migrational capacity and perturbations in chromatin condensation which could oppose radiation. Based on the displayed adaptions in cellular protein expression and signaling in resistant PDAC cells, pharmacological inhibition of NQO1 and FAK could be suggested to sensitize towards radiation.

Project description:Purpose: Predominant causes of head and neck cancer recurrence after radiotherapy are rapid repopulation, hypoxia, fraction of cancer stem cells and intrinsic radioresistance. Currently, intrinsic radioresistance can only be assessed by ex-vivo colony assays. Besides being time-consuming, colony assays do not identify causes of intrinsic resistance. We aimed to identify a biomarker for intrinsic radioresistance to be used before start of treatment and to reveal biological processes that could be targeted to overcome intrinsic resistance. Experimental design: We analyzed both micro- and messenger RNA expression in a large panel of HNSCC cell lines. Expression was measured on both irradiated and unirradiated samples. Results were validated using modified cell lines and a series of laryngeal cancer patients. Results: MiRs, mRNAs and gene sets that correlated with resistance could be identified from expression data of unirradiated cells. The presence of epithelial to mesenchymal transition (EMT) and low expression of miRs involved in the inhibition of EMT were important radioresistance determinants. This finding was validated in two independent cell line pairs, in which the induction of EMT reduced radiosensitivity. Moreover, low expression of the most important miR (miR-203) was shown to correlate with local disease recurrence after radiotherapy in a series of laryngeal cancer patients. Conclusions: These findings indicate that EMT and low expression of EMT-inhibiting miRs, especially miR-203, measured in pre-treatment material, causes intrinsic radioresistance of HNSCC, which could enable identification and treatment modification of radioresistant tumors. Matched pairs of pre-treatment biopsies of 34 patients treated at The Netherlands Cancer Institute between 2002 and 2010. Patients with T2-3 laryngeal cancers, all treated with radiotherapy alone with a curative intent. The series was designed to be a matched cohort of 17 patients with local recurrences matched with 17 local cures. There were no significant differences between groups with and without local recurrence in age, gender, subsite, T-stage or treatment year.

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FOXM1-mediated regulation of reactive oxygen species and radioresistance in oral squamous cell carcinoma cells

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